The object of this research is to increase our understanding of the mechanism of RNA polymerase by utilizing spectroscopic methods such as circular dichroism, difference spectroscopy and fluorescence. Conformational changes in synthetic polynucleotides and in a cloned promoter-enriched fragment from T7 DNA will be studied as a function of temperature and ionic strength. Conformational changes in the protein upon binding to DNA are also being characterized. Photoaffinity labeling of RNA polymerase by the substrate analog 8-azido ATP is being investigated. The effects of binding the allosteric effector ppGpp to RNA polymerase are being studied by CD. A fluorescein-labeled derivative of RNA polymerase has been shown to undergo substantial quenching of fluorescence upon DNA binding. This derivative will be used for stopped-flow kinetic studies of DNA-RNA polymerase interaction. Steady-state kinetic studies of RNA polymerase will also be pursued to study a possible isomerization reaction detected in the early stages of the reaction.